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Effects of a Bioactive Scaffold Containing a Sustained TGF-β1-Releasing Nanoparticle System on the Migration and Differentiation of Stem Cells from the Apical Papilla
by
Craig D. Bellamy
A thesis submitted in conformity with the requirements for the degree of Master of Science
Faculty of Dentistry, School of Graduate Studies University of Toronto
© Copyright by Craig D. Bellamy 2016
ii
Effects of a Bioactive Scaffold Containing a Sustained TGF-β1-
Releasing Nanoparticle System on the Migration and
Differentiation of Stem Cells from the Apical Papilla
Craig D. Bellamy
Master of Science
Faculty of Dentistry, School of Graduate Studies University of Toronto
2016
Abstract The study aimed to develop and characterize a novel chitosan-based scaffold (CMCS) containing
TGF-β1-releasing chitosan nanoparticles (TGF-β1-CSnp) to enhance migration and
differentiation of SCAP. Part I concerned synthesis and characterization of the scaffold (CMCS)
and TGF-β1-CSnp. Part II examined the effect of sustained TGF-β1 release from scaffold
containing TGF-β1-CSnp on odontogenic differentiation of SCAP. The scaffold demonstrated
properties conducive to cellular activities. Incorporation of TGF-β1 in CSnp allowed sustained
release of TGF-β1 facilitating delivery of a critical concentration of TGF-β1 at the opportune
time. SCAP showed greater viability, migration and biomineralization in the presence of TGF-
β1-CSnp than in the presence of Free TGF-β1. SCAP cultured in TGF-β1-CSnp + scaffold
showed significantly higher dentin matrix protein (DMP)-1 and dentin sialophosphoprotein
(DSPP) signals compared to Free TGF-β1 + scaffold or CSnp + scaffold. These experiments
highlighted the potential of a CMCS based scaffold with growth factor releasing nanoparticles to
promote migration and differentiation of SCAP.
iii
Acknowledgments
The author gratefully acknowledges the contributions of the following individuals:
Dr. Anil Kishen, primary supervisor, for his intelligence, scientific expertise, tireless
encouragement, and constant availability throughout all stages of my research, study execution,
thesis development and endodontic education.
Dr. Calvin Torneck, for sharing his knowledge and unique perspective, which had direct impact
on the study design and final product.
Dr. Anurahda Prakki and Dr. Craig Simmons, for acting as members of the research
committee.
Dr. Suja Shrestha, whose technical proficiency and collaboration was indispensible in allowing
the research project to be completed with precision.
My Family, who supported my pursuit of higher education.
Funding provided from the American Association of Endodontists Foundation and the
Canadian Academy of Endodontics Endowment Fund was instrumental in the timely
completion of this project. With sincerity, I extend my appreciation to the work, contribution,
and generosity of these organizations.
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Table of Contents
Acknowledgments .......................................................................................................................... iii
Table of Contents ........................................................................................................................... iv
List of Figures ................................................................................................................................ vi
Abbreviations ................................................................................................................................ vii
List of Appendices ....................................................................................................................... viii
Chapter 1 Introduction .................................................................................................................... 1
1.1 General Introduction .......................................................................................................... 1
1.2 Objectives and Hypothesis ................................................................................................ 2
1.2.1 Objectives ................................................................................................................... 2
1.2.2 Hypothesis .................................................................................................................. 2
Chapter 2 Literature Review ........................................................................................................... 3
2.1 Apexification ..................................................................................................................... 3
2.1.1 Calcium Hydroxide ..................................................................................................... 3
2.1.2 Mineral Trioxide Aggregate ....................................................................................... 3
2.2 Regenerative Endodontics ................................................................................................. 4
2.2.1 Cell-Based Approach .................................................................................................. 4
2.2.2 Cell-Free Approach .................................................................................................... 5
2.2.3 Scaffolds ..................................................................................................................... 5
2.3 Bioactive Molecules .......................................................................................................... 6
2.3.1 Transforming Growth Factor β-1 ............................................................................... 7
2.4 Carrier systems .................................................................................................................. 7
2.5 Stem Cells .......................................................................................................................... 8
2.5.1 Stem Cells from Apical Papilla (SCAP) ..................................................................... 9
2.6 Cell Homing ....................................................................................................................... 9
Chapter 3 Article ........................................................................................................................... 11
3.1 Abstract ............................................................................................................................ 11
v
3.2 Introduction ...................................................................................................................... 12
3.3 Materials and Methods .................................................................................................... 13
3.3.1 Part I – Synthesis and Characterization of Scaffold and TGF-β1-CSnp .................. 14
3.3.2 Part II – Effect of Scaffold Containing TGF-β1-CSnp on SCAP Differentiation .... 18
3.3.3 Statistical Analysis .................................................................................................... 19
3.4 Results .............................................................................................................................. 19
3.4.1 Characterization of Scaffold ..................................................................................... 19
3.4.2 Characterization of TGF-β1-CSnp ........................................................................... 19
3.4.3 Effect of Scaffold Containing TGF-β1-CSnp on SCAP Differentiation .................. 20
3.5 Discussion ........................................................................................................................ 21
3.6 References ........................................................................................................................ 23
Chapter 4 Discussion, Conclusion and Future Directions ............................................................ 27
4.1 General Discussion .......................................................................................................... 27
4.2 Conclusion ....................................................................................................................... 31
4.3 Future Directions ............................................................................................................. 32
References ..................................................................................................................................... 33
Appendix I .................................................................................................................................... 46
Characterization of scaffold.............................................................................................46
Characterization of TGF-β1-CSnp ..................................................................................47
TGF-β1-induced migration of SCAP ..............................................................................48
Effect of nanoparticles on biomineralization ..................................................................48
Immunofluorescence of DMP-1 and DSPP during odontoblast differentiation .............49
vi
List of Figures
Figure 1: Characterization of scaffold ..........................................................................................46 (A) Scaffold morphology as observed under SEM illustrating porous structure; (B) Scaffold swelling ratio; (C) Scaffold degradation (%) in the presence of lysozyme; (D) FTIR Spectra of CMCS, Gelatin, and the CMCS/gelatin scaffold; (E) SCAP morphology as observed under SEM illustrating favorable response to scaffold.
Figure 2: Characterization of TGF-β1-CSnp ................................................................................47 (A) Cumulative release (ng) of TGF-β1 from TGF-β1-CSnp; (B) Relative viability of SCAP exposed to Free TGF-β1, TGF-β1-CSnp, or CSnp over 1, 3 and 14 days. (C) Representative fluorescent microscopic view after the calcein-AM staining (upper row, magnification 20X) and phase contrast microscopic images (lower row, magnification 40X). Data represent means ± SD (n = 3). *Significant difference between groups, p < 0.001.
Figure 3: ........................................................................................................................................48 (A) TGF-β1-induced migration of SCAP: Cell migration was examined by a Transwell assay. SCAP were cultured in presence of Free TGF-β1, TGF-β1 released from TGF-β1-CSnp incubated for different time periods and CSnp. The lower membrane surfaces were photographed through a microscope at 20X magnification. Migrated cells in each field were counted. *Significant difference between groups, p < 0.028. (B) Effect of nanoparticles on biomineralization: ARS was quantified for biomineralization in SCAP culture for 3 weeks in presence of Free TGF-β1, TGF-β1-CSnp or CSnp. Data are shown as mean OD/mg of total protein. Data represent means ± SD (n = 3). *Significant difference between groups, p < 0.001.
Figure 4: Immunofluorescence of DMP-1 and DSPP during odontoblast differentiation: ...........49 (A) FITC-conjugated antibody was used to detect the localization of the protein (green signal); all samples were counterstained with DAPI (blue signal). (B) Quantification of protein expression by ImageJ software and data are presented as relative mean fluorescence intensity. *Significant difference between groups, p < 0.001.
vii
Abbreviations
BM bioactive molecules
CH calcium hydroxide
CMCS carboxymethyl chitosan
CSnp chitosan nanoparticles
DMP-1 dentin matrix protein-1
DPSC dental pulp stem cells
DSPP dentin sialophosphoprotein
MSC mesenchymal stem cells
MTA mineral trioxide aggregate
SCAP stem cells from apical papilla
TGF-β1 transforming growth factor Beta-1
viii
List of Appendices
Appendix I ................................................................................................................................... 46
1
Chapter 1 Introduction
1.1 General Introduction Bioactive molecules (BM) are critical to mammalian cell growth (1) and thus are
fundamental in the processes of tissue development, function, and healing. The absence
of these BM, or their presence in inappropriate concentrations in a specific tissue
environment can have diverse effects on cell activity, that results in varying biological
effect on the tissue and highlights the complex multifunctional role that BM play in tissue
homeostasis (2). Understanding BM-cell interactions is central to translational
knowledge in tissue regenerative applications including regenerative endodontics.
Studying how and when these interactions occur on a spatial and temporal basis facilitate
this understanding and expedite the clinical application of this knowledge. Current
regenerative endodontic procedures employ tissue engineering principles that exploit
BM-cell interactions (3).
In the past, and perhaps to some degree in the present, apexification has been the
preferred treatment of immature teeth that have become pulpless. However, immature
teeth, subsequent to successful apexification, remain at risk of fracture due to thin root
dentin walls. Recognizing this, endodontists are developing biologically based treatment
strategies to improve the fate of these often-fractured teeth by generating new functional
tissue that they hope will improve the quality of patient’s lives (4, 5).
An alternative to apexification is a regenerative endodontic procedure. However, at
present the outcomes of these procedures remain questionable (6). Different protocols to
regenerate pulp and dentin have been investigated (7, 8) and different materials and
different BM have been used to stimulate and support new tissue growth (9-13).
Molecules of the transforming growth factor (TGF)-β superfamily are involved in the
regulation of cell proliferation, cell differentiation, epithelial-mesenchymal transition,
and embryonic development (14). Employing tissue-engineering principles, scaffolds,
stem cells and bioactive molecules have been used to regenerate neo-tissue at a desired
site over a predictable period of time. Polymeric-based scaffolds have been investigated
and offer only measured success. Thus, no scaffold material has gained universal
2
acceptance. Additionally, augmentation of scaffold bioactivity using BM release to
predictably generate new tissue in dentin pulp engineering has remained a challenge (3).
While no preferred scaffold material exists in regenerative endodontics, the many
positive attributes of a chitosan-based scaffold may make it an optimum material for this
application (15, 16).
Tissue healing and repair of dental pulp and reparative dentin formation, is a result of
distinct BM interaction, which drives cell chemotaxis, proliferation, differentiation, and
extracellular matrix remodeling (17). The current study investigated the potential of a
novel chitosan-based scaffold containing biomolecule (TGF-β1)-releasing CSnp (TGF-
β1-CSnp) to enhance migration and differentiation of SCAP, since cell migration and
differentiation are critical initial steps in the regeneration of the pulp-dentin complex.
1.2 Objectives and Hypothesis
1.2.1 Objectives I. To develop and characterize a chitosan-based scaffold and a novel TGF-β1-releasing
chitosan nanoparticle (CSnp) system.
II. To examine the effect of chitosan scaffold containing TGF-β1-CSnp on migration and
odontogenic differentiation of SCAP.
1.2.2 Hypothesis A bioactive chitosan scaffold containing a sustained TGF-β1-releasing CSnp system will
promote migration and enhance differentiation of SCAP.
3
Chapter 2 Literature Review
2
2.1 Apexification Apexification can be defined as a method to induce a calcified barrier at the apical end of
a root with an open apex or continued apical development of an incompletely formed root
in teeth with necrotic pulps (18). Two materials currently used in this method are
calcium hydroxide (CH) and mineral trioxide aggregate (MTA) (19).
2.1.1 Calcium Hydroxide Calcium hydroxide is a low cost, easily obtained, highly alkaline (pH 12) material formed
by the combination of calcium oxide and water. Introduced to dentistry by Hermann (20),
its application on exposed dental pulp leads to the formation of a hard tissue or calcific
bridge over the exposure (21). Similarly, its application within the root canal results in
hard tissue formation in the root canal (pulpotomy) or at the root apex (apexification).
Additional benefits of CH include neutralization of low pH acid, raising of tissue pH,
stimulation of fibroblasts, as well as antimicrobial effect through various mechanisms
such as membrane lipid peroxidation, protein denaturation, and splitting of microbial
DNA strands (21, 22). However, use of CH in apexification has drawbacks. Multiple
appointments over an extended period of time (months to years) are required to disinfect
the root canal and induce the formation of calcified tissue at the root end where it
facilitates the placement of a root canal filling. In addition, prolonged intracanal use of
CH has been shown to alter the dentin matrix, making the tooth susceptible to fracture
(23-26).
2.1.2 Mineral Trioxide Aggregate Mineral trioxide aggregate (MTA) is a powder consisting of tricalcium silicate, tricalcium
aluminate, tricalcium oxide, bismuth oxide, silicate oxide, tetracalciumalumino ferrite,
and calcium sulphate dehydrates (27). When mixed with moisture, these particles set to
form an alkaline (pH 12) colloidal gel initially, which then solidifies to a hard structure
over approximately four hours. MTA is biocompatible and shows excellent sealing
ability when applied to tooth structure. Additionally, new tissue formation has been
4
shown in direct apposition to MTA in vivo (28). Due to these favourable characteristics,
it has been employed in immediate or ‘one-step’ apexification procedures. Apically
placed MTA establishes a root-end ‘plug’ by mechanical means against which root filling
material can be packed, while circumventing some of the drawbacks associated with CH
apexification (28, 29). Unfortunately, even when MTA apexification is successful, like
CH, root width is not increased and tooth susceptibility to fracture remains (30).
Children between the ages of 7 to 15 have a high incidence of dental trauma (31). This is
an age when most permanent teeth are incompletely formed and when jaw bone
development makes tooth replacement with an osseo-integrated implant contraindicated
(32, 33). It has also been reported that up to 50% of traumatized teeth may be diagnosed
with pulp necrosis (34). Traumatized teeth with immature roots that are treated
endodontically show a cervical fracture incidence between 28%-77% over four years (35).
Thus, it has been suggested that a treatment approach that increases the tooth root width
and reduces fracture susceptibility, is preferable (36).
2.2 Regenerative Endodontics Regenerative endodontics refers to biologically based procedures designed to
physiologically replace damaged or undeveloped tooth structure, including dentin and
root structures, as well as cells of the pulp-dentin complex (18). These procedures
employ classic tissue engineering techniques comprised of stem cells, scaffolds, and
bioactive growth factors (3). Two such procedures involve cell-based and cell-free
approaches.
2.2.1 Cell-Based Approach A cell-based tissue engineering approach involves the application of cells exogenously to
the site where tissue regeneration is desired. This approach presents several challenges.
Allogeneic cell transplants can undergo immune rejection and lead to heterotopic tissue
formation. Additionally, the sourcing and expansion of cell populations appropriate for
this application can be time-consuming and costly (37). Furthermore, transplanted cells
are particularly vulnerable to their environment, and optimizing the balance between
delivery and consumption of oxygen and nutrients is difficult (38). Selection of cell type
5
and concentration to be transplanted needs careful consideration as this has direct impact
on the nutrient availability and metabolism of these cells, which can subsequently affect
the overall performance of the implanted materials (39).
2.2.2 Cell-Free Approach A cell-free tissue engineering approach involves recruitment or homing of endogenous
cells to the desired site of regeneration. This approach may overcome some of the
challenges associated with the cell-based technique previously mentioned (40). Different
protocols for cell-free dentin-pulp tissue regeneration have been studied for many years
(7, 8, 41-43) as have the different materials used to support cell growth and deliver
appropriate bioactive molecules to the site of regeneration (9-13, 15, 16, 44-68).
Employing a conducive scaffold in regenerative endodontic applications is critical.
Several polymeric materials have been used as scaffolds either alone or in combination,
but optimizing a scaffold material together with controlled delivery of bioactive
molecules has remained a challenge.
2.2.3 Scaffolds Scaffolds are materials that serve a structurally supportive role for developing tissue in
regenerative procedures. Additionally, they provide a surface for cell attachment and
may act as a vehicle for bioactive molecule delivery. Materials used for scaffolds in
regenerative applications should be biocompatible, substantive, tunable, porous, reactive,
and biodegradable (3). These materials include natural polymers like polysaccharides
and extra-cellular matrix (ECM); synthetic biodegradable polymers such as polyesters
(e.g. PGA, PLA, PCL) and polyurethanes; bioceramics (e.g. calcium phosphates,
hydroxyapatite, tricalcium phosphate, bioactive glasses); and hydrogels (e.g. collagen,
alginate, chitosan). Blood clots and platelet-rich plasma (PRP) have also been used (69).
2.2.3.1 Chitosan While no ‘gold standard’ scaffold material has been established in regenerative
endodontics, chitosan-based scaffolds may be best suited for dentin-pulp engineering, as
chitosan possesses many desirable attributes for this application. Chitosan is the N-
deacetylated derivative of chitin. Chitin is a naturally abundant polysaccharide, and the
6
supporting material of crustaceans and insects; it functions naturally as a structural
polysaccharide (15). Chitosan is a bioactive biopolymer with a cationic charge that offers
substantive benefits when interacting with hard tissues including root dentin (70). It can
be customized to form scaffolds that swell and adapt well to recipient sites (68). It has
alterable porosity to permit transport of nutrients, biomolecules, and toxic cellular
products. Its structural strength and dissolution characteristics can be altered. It is non-
toxic, antibacterial, angiogenic, resistant to endotoxin and inhibits collagen degradation
(16, 55, 68-83). Several studies have reported many favorable characteristics of different
forms of chitosan that match those of an ideal scaffold, highlighting chitosan’s viability
as a scaffold material in tissue regeneration (45, 68, 70, 73, 76, 78-82). Carboxymethyl
chitosan (CMCS), a water-soluble derivative of chitosan, has displayed several beneficial
characteristics when used as a scaffold (84), and as a surface-modifier of dentin matrix to
enhance antibacterial efficacy and ultrastructural characteristics (83, 85).
2.2.3.2 Gelatin Gelatin is a natural polymer obtained by a controlled acid or alkaline hydrolysis of native
collagen (86). Due to its excellent biocompatibility, biodegradability, and nontoxicity,
gelatin has been employed in medicine and pharmaceutics as a material for controlled
drug release (87). Gelatin alone does not offer appropriate strength or substantivity when
used in tissue regenerative applications. However, when combined with chitosan, gelatin
produces improved mechanical properties and swelling capacity of the scaffolds in an
aqueous environment (88).
2.3 Bioactive Molecules Bioactive molecules is an umbrella term referring to a diverse group of molecules
encompassing growth factors, chemokines, cytokines, extracellular matrix molecules, and
bioactive peptides. Growth factors can be defined as extracellular signalling proteins that
are involved in cell to cell communications (89). Some concerns regarding the
application of BM in regenerative procedures include the potential for their uncontrolled
release, toxicity, short half-life, and possible lack of effect. The release of BM from
polymeric materials can occur over an extended period of time without loss of bioactivity
(90-92). The culture time for complete osteogenic differentiation of mesenchymal stem
7
cells in vitro has been reported to take place over 3 weeks, but this process occurs faster
in the presence of certain BM (92-95), highlighting the importance of evaluating the
beneficial effects of sustained release of BM in this study. One of the most common
strategies to achieve sustained release of BM is to incorporate them into polymeric
biomaterials.
2.3.1 Transforming Growth Factor β-1 Several BM have been evaluated in tissue engineering (96, 97) and many different BM
have been reported to be sequestered within the dentin matrix (98). The current literature
indicates that the transforming growth factor (TGF)- β superfamily is one of the most
important growth factors in dentin-pulp regeneration (99, 100). The TGF-β superfamily
regulates cell proliferation, cell differentiation, the epithelial-mesenchymal transition and
embryonic development (14). An in vitro human tooth model based investigation
suggested TGF-β1 to have direct involvement in the regulation of cell proliferation,
migration and extracellular matrix synthesis in the human dental pulp and in the repair
process occurring after tooth injury (101). It has been shown that TGF-β1 localizes in the
dentin matrix, and implantation of isolated dentin proteins and dentin matrix (102, 103)
in dental pulp induced odontoblast-like differentiation. TGF-β1 cell signalling may occur
through TGF-β receptor internalization into the cell via clathrin-coated vesicle
endocytosis as well as via membrane caveolae (104). Clathrin-mediated endocytosis has
been shown to promote TGF-β-induced Smad activation and transcriptional responses.
Caveolae are regarded as signaling centers for G protein-coupled receptors and tyrosine
kinase receptors, but they are indicated to facilitate the degradation of TGF-β receptors
and therefore, turnoff of TGF-β signaling (104). Evidence also indicates that TGF-β
signaling can take place on the cell plasma membrane (105).
2.4 Carrier systems Therapeutic applications of growth factors pose a major challenge clinically due to their
short half-life and rapid diffusion into the surrounding medium and this may have
deleterious effects in vivo (106). Bioactive molecules can be introduced into many tissue
engineering systems by various methods, which include adding directly to the medium,
by genetically engineering cells to overexpress them, and by constructing polymeric
8
systems that allow their controlled release (107). Challenges of biomolecule delivery
previously mentioned may be overcome if polymeric carrier systems are engineered to
deliver bioactive molecules over time and with a degree of spatial control. This provides
an effective concentration of active BM for endogenous cells since spatiotemporal
expression of BM has been shown in odontogenesis in vivo (108). Examples of BM
carriers include the scaffold itself, microparticles, or nanoparticles. Khil et al. (109)
designed a porous chitosan scaffold, containing chitosan microspheres loaded with TGF-
β1, to enhance chondrogenesis. They demonstrated that the scaffolds containing the
loaded chitosan microspheres significantly increased the cell proliferation and production
of extracellular matrix (ECM). A similar approach using chitosan-based materials has
been reported (107), where three dimensional collagen/chitosan/glycosaminoglycan
scaffolds were seeded with rabbit chondrocytes and combined with TGF-β1-loaded
chitosan microspheres. This allowed for an evaluation of the effect of released TGF-β1
on the chondrogenic potential of rabbit chondrocytes in such a system. Nanoparticles are
very small particles, approximately 1-100nm in size (110). Functionalized chitosan
nanoparticles have been shown to be effective in the temporal-controlled delivery of BM
to an odontogenic line of stem cells (111, 112) and its use has enhanced cell proliferation
and differentiation in dentin-pulp engineering (113, 114).
2.5 Stem Cells Stem cells are unspecialized, immature cells with the potential to develop into many
different cell lineages via differentiation and hence they are useful in regenerative
strategies (115). The Mesenchymal and Tissue Stem Cell Committee of the International
Society for Cellular Therapy (ISCT) proposed minimal criteria to define human MSC
(116). First, MSC must be plastic-adherent when maintained in standard culture
conditions. Second, MSC must express CD105, CD73 and CD90, and lack expression of
CD45, CD34, and other negative markers. Third, MSC must differentiate to osteoblasts,
adipocytes and chondroblasts in vitro. Various tissue-specific stem cell populations have
been described including those pertaining to oral tissues (117, 118). Some of these stem
cells include salivary gland stem cells (SGSC), oral epithelial stem cells (OESC),
periosteal derived stem cells (PSC), dental follicle progenitor cells (DFPC), dental pulp
9
stem cells (DPSC), stem cells from human exfoliating deciduous teeth (SHED),
periodontal ligament stem cells (PDLSC), inflammatory periapical progenitor cells
(IPPC), bone marrow stem cells (BMSC), and stem cells from the apical papilla (SCAP)
(119).
2.5.1 Stem Cells from Apical Papilla (SCAP) Stem cells from the apical papilla possess several characteristics that make them an
excellent choice for studying dentin-pulp tissue regeneration. First characterized in 2006,
SCAP are mesenchymal stem cells in the apical papilla of developing teeth that can
differentiate into odontoblast-like cells forming dentin (120). It has been shown that
SCAP play a role in root development (121). Furthermore, SCAP have demonstrated an
elevated tissue regeneration capacity, higher telomerase activity, and improved migration
capacity when compared to DPSC from the same tooth (120). SCAP have been shown to
regenerate vascularized human dental pulp-like tissues suggesting their potential as a
source of primary odontoblasts (122). Previous investigators have demonstrated that
SCAP show a significantly higher proliferation rate and mineralization potential than
DPSC (123). In addition, SCAP are derived from a very accessible tissue resource and
are relatively easy to expand in vitro so as to provide enough cells for potential clinical
applications. Isolated SCAP grown in cultures can undergo dentinogenic differentiation
when stimulated with biomolecules and have also been shown to play a potential role in
continued root formation of immature pulpless teeth (62). Studies have shown that SCAP
cells remain viable and proliferate after exposure of dentin to EDTA, NaOCl, and CH,
which are typical irrigants and medicaments used in endodontics (124, 125).
2.6 Cell Homing Cell homing has been regarded as a process by which hematopoietic stem cells exit from
blood vessels (trans-endothelialization) and migrate to a site (126). In tissue engineering,
cell homing is the active recruitment of endogenous cells, including stem/progenitor cells
into the anatomic site of regeneration. Cell homing techniques have been used in the
regeneration of dental tissues (127, 128). Chemotaxis-induced cell homing has been
shown to result in re-cellularization and revascularization in the endodontically prepared
root canal in vivo (97, 129). Platelet rich plasma (PRP) has been a popular focus of
10
investigation as a scaffold as well as a homing tool due to its rich supply of autologous
BM (13, 60, 130-133). However, PRP preparation is cumbersome and control of platelet
concentration and therefore control of BM concentration is unpredictable and may lead to
a detrimental effect on stem cells (134). Since, as mentioned, BM have been shown to be
effective in recruiting and encouraging odontogenic differentiation of stem/progenitor
cells, the use of specific BM may prove to be essential to the successful evolution of
regenerative protocols in the management of immature permanent teeth with necrotic
pulps.
11
Chapter 3 Article
3
3.1 Abstract Introduction: This two-part study hypothesized that novel modified chitosan-based
scaffold containing a sustained transforming growth factor (TGF)-β1-releasing
nanoparticle system (TGF-β1-CSnp) will promote migration and enhance differentiation
of stem cells from the apical papilla (SCAP) as compared to Free TGF -β1 + scaffold,
and the scaffold alone. Methods: Part I describes the synthesis and characterization of a
carboxymethyl chitosan (CMCS) based scaffold and the characterization of the TGF-β1-
CSnp. Part II examines the effect of sustained TGF-β1 release from scaffold containing
TGF-β1-CSnp on SCAP vitality, migration, and odontogenic differentiation. Results:
The scaffold demonstrated acceptable biocompatibility and physical properties conducive
to the maintenance of cellular function. The incorporation of TGF-β1 in CSnp allowed
for sustained release of a critical concentration of TGF-β1 to SCAP over the term of the
study. Except for an initial period of increased SCAP migration noted with Free TGF-β1
+ scaffold, SCAP showed greater viability, migration, biomineralization, and
odontogenic potential, with TGF-β1-CSnp + scaffold than with Free TGF-β1 + scaffold
or CSnp + scaffold. Conclusions: These experiments highlighted the potential of a
CMCS based scaffold with engineered growth factor releasing nanoparticles to promote
migration and differentiation of SCAP over time, and may have direct application to
improve current endodontic regenerative protocols.
Key words: Stem cells from apical papilla, chitosan nanoparticles, transforming growth
factor-β1, cell migration, odontogenic differentiation, regenerative endodontic procedures
12
3.2 Introduction Tooth retention has mechanical, biological, and psychological benefits that include
chewing efficiency, speech, immune-competence, and self-confidence (4, 5). Endodontic
treatment of mature permanent teeth with apical periodontitis has proven to be highly
successful in retaining teeth that at one time were routinely extracted (135). Treatment of
immature permanent teeth with apical periodontitis has proven to be more challenging
and less predictable. Currently, the treatment strategies used in the management of
immature teeth with necrotic pulps include apexification using calcium hydroxide,
placement of an apical ‘plug’ of MTA, and regenerative endodontic procedures.
Apexification requires multiple appointments over an extended period of time to disinfect
the root canal, and induce formation of calcified tissue at the root end to facilitate
placement of a root canal filling. An apically placed MTA ‘plug’ attempts to achieve the
same end mechanically but usually requires less treatment appointments. However both
of these treatment strategies fail to produce an increase in the thickness of the root canal
wall even when successful, leaving the root susceptible to fracture over time (30).
Regenerative endodontics is a viable treatment alternative, but the predictability for a
desirable treatment outcome remains questionable (6). Different protocols to regenerate
pulp and dentin have been investigated (7, 8) and different materials and different
bioactive molecules (BM) have been used to stimulate and support new tissue growth (9-
13). The current literature indicates that members of the transforming growth factor
(TGF)- β superfamily are important growth factors in dentin-pulp regeneration (99, 100)
and have played a role in the regulation of cell proliferation, cell differentiation,
epithelial-mesenchymal transition, and embryonic development (14). A scaffold-based
regenerative protocol uses an engineered substrate to attract a stem cell population to a
specific site. In regenerative endodontics, the scaffold should ideally possess tissue-
forming qualities that are expressed in suitable concentrations at opportune times.
Several polymeric-based scaffolds have been used in regenerative endodontics with
measured success, however, optimizing their bioactivity with temporal-controlled BM to
enhance new tissue formation has remained a challenge (3). While no preferred scaffold
13
material exists, a chitosan-based scaffold may prove to be preferable since chitosan
exhibits many properties favorable to new pulp and dentin formation.
Chitosan is a ubiquitous bioactive biopolymer with a cationic charge that offers
substantive benefits when interacting with hard tissues including root dentin (70). It can
be customized to form scaffolds that swell and adapt well to recipient sites (68) and it has
alterable porosity to permit transport of nutrients, biomolecules, and toxic cellular
products. Its structural strength and dissolution characteristics can be altered, and it is
non-toxic, antibacterial, angiogenic, and resistant to endotoxin and bacterial degradation
(16, 70, 83).
Carboxymethyl chitosan (CMCS), a water-soluble derivative of chitosan, has displayed
several favorable characteristics when used as a scaffold (84), and as a surface-modifier
of dentin matrix since it has the ability to enhance antibacterial efficacy and stabilize the
dentin matrix (83, 85). Functionalized CSnp have shown effectiveness in the temporal
(time)-controlled delivery of BM to an odontogenic line of stem cells (111, 112), and the
ability to enhance cell proliferation and differentiation in tissue regeneration (113, 114).
Since cell migration and differentiation is a critical initial step in the regeneration of pulp
and dentin, the current study investigated the potential of a novel CMCS scaffold
containing TGF-β1-releasing CSnp (TGF-β1-CSnp) to enhance migration and
differentiation of SCAP.
3.3 Materials and Methods In the first part of this study we assessed the physical and chemical characteristics and
biocompatibility of a synthesized CMCS-based scaffold and TGF-β1-CSnp. In the
second part we assessed the effect of sustained release of TGF-β1 from the scaffold
containing TGF-β1-CSnp on SCAP migration and odontogenic differentiation relative to
the effect of Free TGF-β1 + scaffold, unloaded chitosan nanoparticles + scaffold and the
scaffold alone. All chemicals used in the study were of analytical grade and purchased
from Sigma-Aldrich Inc. (St Louis, MO) unless noted otherwise. TGF-β1 (catalog #
ab50036) and the corresponding ELISA kit were purchased from Abcam Inc. (Toronto,
ON, Canada).
14
3.3.1 Part I – Synthesis and Characterization of Scaffold and TGF-β1-CSnp
3.3.1.1 Synthesis and Characterization of Scaffold To prepare the scaffold a 2% (1.5 mL) CMCS solution was added to 15% (1.5 mL) warm
gelatin solution and mixed in a 35 mm tissue culture plate to produce a gel. The gel was
kept at -20°C overnight, and lyophilized at -60°C for 24 h. The dry scaffolds were
immersed in a 2 mL solution of 40% ethanol containing 50 mM methane sulfonic acid
(MES) (pH 5.0), 33 mM N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride
(EDC) and 8 mM N-hydroxysuccinimide (NHS) for 10 h at room temperature. After
neutralizing with 0.1 M Na2HPO4 (pH 9.1) for 1 h, the scaffolds were washed and
lyophilized again for 24 h before testing.
3.3.1.1.1 Porosity Analysis Three scaffolds in each group to be tested later were weighed before and after immersion
in absolute ethanol overnight. Porosity of the scaffolds was calculated using the
following formula (136);
Interval porosity (%) = (! ! !!)!!!!!!(!!!!!)!!
× 100
Where, 𝑊 = wet weight, 𝑊! = dry weight, 𝜌!= average density of CMCS and gelatin
(0.95 g/mL), 𝜌! = density of anhydrous alcohol (0.79 g/mL).
3.3.1.1.2 Swelling Analysis Dry weight of each scaffold was recorded before and after immersion in PBS at 37°C.
Wet scaffolds were removed at regular time intervals, gently blotted with filter paper and
weighed. The following formula was used to calculate the swelling ratio (137);
Swelling ratio (%) = !!"#! !!"#
!!"# × 100
Where, 𝑊!"# = initial dry weight, 𝑊!"#= weight after swelling
15
3.3.1.1.3 Biodegradation Analysis Scaffolds (20 ± 1 mg) were sterilized in 100% ethanol overnight and washed with PBS.
Sterilized scaffolds were immersed in 2 mL of PBS containing 10 mg/L lysozyme (Fisher
Bioreagents; catalog # BP535-1; Activity = 24,200 units/mg), and incubated at 37°C.
Lysozyme was refreshed every 3 days. Scaffolds were removed from the lysozyme
solution at different time intervals, washed, kept at -20°C for 2 h, lyophilized, then
measured to determine dry weight. Degree of degradation was calculated using the
following formula;
Degradation (%) = !!! !!
!! × 100
Where, 𝑊! = initial dry weight, 𝑊! = dry weight after degradation
3.3.1.1.4 Chemical Characterization Chemical characterization of the scaffold was carried out by Fourier transform infrared
(FTIR) spectroscopy. Transmission spectra of powdered scaffold mixed with potassium
bromide in a 1:10 ratio (w/w) were recorded using a FTIR Spectrophotometer (Paragon
500, Perkin Elmer, Waltham, MA, USA) from 4000 to 400 cm-1 with 16 scans and 4 cm-1
resolution.
3.3.1.1.5 SCAP Viability in Scaffold A characterized SCAP cell line was used in the study (138). Cells were cultured in α-
minimum essential medium (MEM) supplemented with 10% fetal bovine serum, 2 mM
L-glutamine and 100-units/mL antibiotic. Sub-confluent SCAP of passage 3 were
detached using trypsin/EDTA and re-suspended at an appropriate concentration. Cultures
were kept at 37°C in a humidified incubator with an atmosphere of 5% CO2. (Thermo
Scientific, Waltham, MA, USA). Dry scaffolds (4 mm diameter) were immersed in
100% ethanol overnight in 96-well plates, washed with PBS, then immersed in culture
medium overnight. SCAP (2X104) were cultured in the scaffolds for 1 week. SCAP
viability was quantitatively analyzed using a Live/Dead viability/cytotoxicity assay kit
(Invitrogen, Carlsbad, CA). Indicator dyes (1µM Calcein-AM and 2µM EthD-1 in PBS,
Molecular Probes, Inc. Eugene, OR, USA) were added and the fluorescence signal
(excitation: 485/530 nm; emission: 530/645 nm) from the resulting solution was obtained.
16
The background signals (scaffolds without cells) were subtracted before calculating the
cell viability. Subsequently, scaffolds were fixed overnight with 2.5% glutaraldehyde
(4°C) and dehydrated in graded ethanol and hexamethylenedisilane for scanning electron
microscopy (Hitachi S-2500, Japan).
3.3.1.2 Synthesis and Characterization of TGF-β1-CSnp TGF-β1-CSnp was synthesized by the adsorption method previously described (113).
Briefly, freshly prepared CSnp was re-suspended in 10 mL PBS, to which 2 µg of TGF-
β1 dissolved in 400 µL of 4 mM HCl was added. The mixture was stirred at 4°C
overnight. Nanoparticles were washed with isopropyl alcohol, then water and lyophilized
before use.
3.3.1.2.1 Particle Size and Charge The size/charge of nanoparticles was determined by transmission electron microscopy
(CM12 Philips, The Netherlands) and dynamic light scattering using a Zetasizer (Nano
series, Nano-ZS90, Malvern, UK), respectively.
3.3.1.2.2 Release Kinetics Release kinetics of TGF-β1-CSnp were studied, following manufacturer instructions in
the ELISA kit. Briefly, the amount of TGF-β1 released by 10 mg of TGF-β1-CSnp in 1
mL PBS (pH 7.4) at 37°C was measured by analyzing the supernatant of the
centrifuged mixture. Samples were centrifuged, and at predetermined time points, the
supernatant was collected and immediately frozen. Frozen supernatant was then allowed
to thaw and the amount of TGF-β1 in the mixture was quantified.
3.3.1.2.3 SCAP Viability SCAP viability was quantitatively analyzed with a resazurin-based assay using a viability
assay kit (Promega, USA). Approximately 2X104 SCAP were seeded into 96-well plates
and incubated at 37°C for 24 h. Cells were treated with a nanoparticle suspension (300
µg/mL) or Free TGF-β1 (5 ng/mL) in a culture medium and cultured for 14 days. SCAP
cultured in the absence of nanoparticles or BM was used as a control. Groups tested were:
(a) no nanoparticles, (b) Free TGF-β1, (c) TGF-β1-CSnp and (d) CSnp. Cell survival was
determined by adding 40 µL of reagent directly into the cell culture. Fluorescence was
17
measured after incubation (37°C) for 4 h. SCAP stained with calcein-AM was examined
by fluorescent microscopy (Vert.A1, Carl Zeiss, Germany) and phase contrast
microscopy to assess cell morphology.
3.3.1.2.4 Migration Assay SCAP migration was assessed using a 24-well Transwell migration assay (6.5 mm
diameter; 8µm pore size polycarbonate membrane, Corning, Lowell, MA). SCAP
(1X104) in supplement free α-MEM were seeded into the upper chambers and allowed to
attach for 4 h before exposure to test conditions. The lower chambers of the multi-well
system contained test solutions conditioned with (a) no nanoparticles, (b) Free TGF-β1
(10 ng/mL) + scaffold, (c) TGF-β1-CSnp (2 mg/mL) + scaffold or (d) CSnp (2 mg/mL) +
scaffold for pre-determined time points. Cells were cultured at 37°C in 5% CO2. After
incubation for 24 h, cells were fixed in 4% paraformaldehyde for 10 min, and incubated
with 25 µg/ml 4’,6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature.
Non-migrated cells on the upper surface of the membrane were carefully removed and
cells that had migrated through the membrane to the lower surface of the well were
imaged using a fluorescence microscope. Migrated cells from 14 fields taken from 3
wells of each group were counted to assess cell migration.
3.3.1.2.5 Biomineralization Assay SCAP (5X104) were grown to confluence in 6-well culture plates in standard culture
medium. Standard medium was then replaced with a mineralizing medium containing 50
µg/mL ascorbic acid, 10 mM β-glycerolphosphate and 1.8 mM KH2PO4 in standard
medium (113). Free TGF-β1 (3 ng/mL), TGF-β1-CSnp (500 µg/mL) or CSnp (500
µg/mL) was then added to the mineralizing medium and SCAP for 21 days at 37°C and
5% CO2. Biomineralization potential was assessed by staining with alizarin red stain
(ARS). In brief, cells were washed 3 times with PBS and fixed in 10% normal buffered
formalin for 30 min at room temperature. After additional washing, they were incubated
in 2% ARS (pH 4.2) for 20 min at room temperature with gentle agitation, followed by
washing and air-drying. Stained cells were incubated with a 10% cetylpyridinium
chloride in 10 mM sodium phosphate buffer for 30 min to elute calcium-bound stain.
Supernatant was collected, and the optical density determined at 560 nm. Mineralized
18
nodule formation was represented as OD per mg of total cellular protein, determined by a
BCA protein assay kit (Sigma-Aldrich, St Louis, MO, USA). Background staining caused
by CSnp without cells was subtracted from the experimental data to provide a net score.
3.3.2 Part II – Effect of Scaffold Containing TGF-β1-CSnp on SCAP Differentiation
3.3.2.1 Preparation of Scaffold Containing Nanoparticles Solution containing Free TGF-β1 (9 ng), TGF-β1-CSnp (1.5 mg) or CSnp (1.5 mg) in
500 µL ethanol was carefully introduced into scaffold 35 mm in diameter and
immediately lyophilized to prepare the scaffolds containing nanoparticles for immediate
use (107).
3.3.2.2 Immunofluorescence Analysis Scaffolds containing Free TGF-β1, TGF-β1-CSnp or CSnp were placed in a 6-well plate
and seeded with SCAP (5X104 cells/well) in mineralizing medium. Test groups included
(a) Free TGF-β1 + scaffold, (b) TGF-β1-CSnp + scaffold, (c) CSnp + scaffold and (d)
scaffold. After 14 d of culture at 37°C, a 20 mm diameter sample from each group was
frozen in Tissue-Tek O.C.T. Compound (Electron Microscopy Sciences, Hatfield, USA)
in liquid nitrogen and sectioned at 25-30 mm in a cryostat at -20°C. Sections were fixed
in 4% paraformaldehyde containing 0.1% Triton-X 100 at 4°C for 30 min, then washed
and blocked with 2.5% BSA for 30 min. Sections were then incubated with mouse anti-
DMP-1 antibody (catalog# sc-73633; Santa Cruz Biotechnologies, CA, USA) and mouse
anti-DSPP antibody (catalog# sc-73632; Santa Cruz Biotechnologies, CA, USA)
antibody, diluted 1:50 in blocking reagent at 37°C for 2 h. After several washes in PBS
/Tween 20, specimens were incubated with secondary antibody from goat anti-mouse IgG
FITC conjugate (catalog# sc-2010; Santa Cruz Biotechnologies, CA, USA) and diluted
1:1500 in PBS at 37°C for 1 h. After rinsing, specimens were counterstained in DAPI and
examined under confocal laser scanning microscopy (Leica Microsystems, Illinois, USA).
Quantitation of proteins expression was carried out using ImageJ software (NIH,
Bethesda, MA, USA) to calculate relative mean fluorescence intensity.
19
3.3.3 Statistical Analysis Data obtained from this study are expressed as mean ± standard deviation. Statistical
analyses were performed using one-way analysis of variance (ANOVA) and post hoc
Tukey test. A P value <0.05 was considered statistically significant.
3.4 Results
3.4.1 Characterization of Scaffold
3.4.1.1 Physicochemical Properties: The ultrastructure of the scaffold showed porous structure (Fig. 1A), with an interval
porosity of 78.3 ± 1.8%. The swelling ratio was calculated to be 763.5 ± 164% (by
weight) after 24 h (Fig. 1B). Degradation study showed that 40 ± 8.4% of the scaffold
was degraded in 3 weeks (Fig. 1C). FTIR spectra displayed presence of cross-linked
CMCS and gelatin in the scaffold (Fig. 1D). The band corresponding to N-H stretching
had shifted to a lower wavelength (3270 cm-1), when compared to CMCS (3350 cm-1)
and gelatin (3396 cm-1), and was slightly broadened. The broadening of the band at 3270
cm-1 of the cross-linked samples was due to OH vibration (H-bonded/non-bonded) from
the CMCS molecules. The formation of amide linkage between amino groups of CMCS
and carboxyl groups of gelatin was confirmed by the shift of the amide II (1514 and 1426
cm-1) and carbonyl bands (1628 cm-1). The band at 1232 cm-1 corresponded to amide III.
The absorption band at 1331 cm-1 was thought to be due to symmetric stretching
vibration of –COOH in the CMCS. Bands at 1110 cm-1 and at 1054 cm-1were
characteristic for -C-O- and –C-O-C- in the CMCS.
3.4.1.2 Effect of Scaffold on SCAP Viability Relative viability of SCAP in the presence of scaffold was 81.2 ± 2% at day 1 and 84.4%
at day 7. SCAP morphology appeared normal under SEM (Fig. 1E).
3.4.2 Characterization of TGF-β1-CSnp
3.4.2.1 Physicochemical Properties Size of TGF-β1-CSnp was 58.9±12 nm and charge was +19.1±1.4. Figure 2A shows the
cumulative release (ng) of TGF-β1 from TGF-β1-CSnp in a sustained manner for up to
20
28 days.
3.4.2.2 Effect on SCAP Viability SCAP were viable in the presence of TGF-β1-CSnp as shown in Figure 2B. A
statistically significant (p<0.001) increase in viability was shown at days 3 and 14 in the
TGF-β1-CSnp group. Viability assessment was further supported by cell morphology as
observed by phase contrast microscopy (Fig. 2C).
3.4.2.3 Effect on SCAP Migration At day 1 SCAP migration with Free TGF-β1 + scaffold was significantly higher
(p<0.001) than that seen with TGF-β1-CSnp + scaffold (Fig. 3A). At day 7 cell
migration with Free TGF-β1 + scaffold was significantly reduced by 80% (p<0.001) and
showed only a 10% increase after 14 and 21 days. There was no statistical difference in
cell migration in the TGF-β1-CSnp + scaffold group between 1 and 7 days, and a 10%
increase in cell migration after 14 and 21 days as compared to that seen at 7 days. There
was a statistically significant difference (p<0.028) in cell migration between Free TGF-
β1 + scaffold and TGF-β1-CSnp + scaffold group after 21 days.
3.4.2.4 Effect on Biomineralization Biomineralization was significantly higher (p<0.001) in the TGF-β1-CSnp + scaffold
group after 7 days than that seen in Free TGF-β1 + scaffold and CSnp + scaffold groups
(Fig. 3B). Biomineralization was also higher in the TGF-β1-CSnp + scaffold group than
the Free TGF-β1 + scaffold or CSnp + scaffold groups after 14 day. No significant
difference between groups was seen at 21 days.
3.4.3 Effect of Scaffold Containing TGF-β1-CSnp on SCAP Differentiation
3.4.3.1 Immunofluorescence analysis Signals for DMP-1 and DSPP were significantly (p<0.001) higher in the TGF-β1-CSnp +
scaffold group than in the Free TGF-β1 + scaffold group (Fig. 4A and 4B). There were
markedly lower signals for both DMP-1 and DSPP in CSnp + scaffold and scaffold only
groups (Fig. 4A and 4B). Immunofluorescence analysis indicated a higher level of SCAP
21
differentiation in the scaffold and a more uniform distribution of the cells in TGF-β1-
CSnp + scaffold group, when compared to other groups.
3.5 Discussion A scaffold capable of attracting stem cells and delivering BM in a temporal-controlled
manner is essential for optimizing tissue engineering. In this study, we described a
CMCS scaffold containing controlled TGF-β1-releasing CSnp that may have practical
application to regenerative endodontics. The engineered matrix/nanoparticle scaffold
mimicked dental extracellular matrix, possessed high porosity, and significantly
enhanced SCAP migration and differentiation.
Scaffolds fabricated by cross-linking gelatin with chitosan derivatives produced
improved mechanical properties and increased swelling capacity (137). Increases in
swelling capacity of the scaffold may be desirable for endodontic applications to improve
scaffold substantivity and adaptation to the root canal walls after implantation.
Favorable scaffold-cell interaction, as observed through SEM in our study, infers that the
CMCS scaffold used in this study was compatible with SCAP. SCAP are ecto-
mesenchymal stem cells from the apical papilla of developing human teeth and represent
a population of cells that can migrate to the root canal wall and participate in tissue
regeneration or organized repair. SCAP has demonstrated increased migration, tissue
regeneration capacity, and telomerase activity, when compared to dental pulp stem cells
from the same tooth (120). It has also been suggested that SCAP may be better equipped
to survive and maintain their potential for differentiation in adverse, low oxygen
conditions (119).
TGF-β1 is a BM released from cells and incorporated into dentin and other non-
collagenous proteins. It has been identified as a promoter of odontogenic differentiation
(103). However, availability of this molecule and other BM sequestered in dentin matrix
may not present in an optimum concentration or for a duration necessary to satisfy stem
cell requirements in tissue regeneration (139). In the environment of a pulpless root
canal, it is crucial that timely release of an optimum concentration of growth factors be
available for cells to survive, migrate and differentiate (140). The importance of sustained
22
release of BM for cell migration and differentiation has previously been shown (113).
Different concentrations of TGF-β1 were employed in this study since it has been shown
that TGF-β1 can effectively promote or inhibit cell activity depending on the available
concentration (107, 141). The significant increase in SCAP viability compared to the
same concentration of non-loaded CSnp or Free TGF-β1 was attributed to the release of
bioactive TGF-β1 in a controlled manner over time. The potential to provide such an
environment was demonstrated in the TGF-β1-CSnp + scaffold group where SCAP
appeared to survive, migrate, and differentiate better than in the other treatment groups.
TGF-β1 is known to modulate many cell activities including cell migration (140).
Significantly higher cell migration after 1 day in the Free TGF-β1 + scaffold group
compared with the TGF-β1-CSnp + scaffold group was expected since all of the TGF-β1
became available to the cells immediately when the TGF-β1 was in free form. After day
7, cell migration in the Free TGF-β1 + scaffold group decreased significantly, which
reflected a significant loss of bioactivity. In contrast, sustained release of TGF-β1 from
TGF-β1-CSnp + scaffold group led to sustained cell migration over time. This was an
important finding in our study, as it showed the ability of the bioactive scaffold
containing TGF-β1-CSnp to address the issue of the short half-life of TGF-β1 (two to
three minutes) when it is used in free form in regenerative procedures (142).
Enhanced biomineralization of SCAP in the presence of TGF-β1-CSnp + scaffold was
also seen and can be attributed to the controlled release of TGF-β1 during the longer
periods of observation, as were the higher levels of DMP-1 and DSPP expression. The
immunofluorescent images also demonstrated more uniform distribution of the cells in
the TGF-β1-CSnp + scaffold group, when compared to other experimental groups
indicating that TGF-β1 release homogenously from the CSnp dispersed in the scaffold.
23
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27
Chapter 4 Discussion, Conclusion and Future Directions
4
4.1 General Discussion A scaffold capable of attracting stem cells and delivering BM in a temporal-controlled
manner is essential for optimizing tissue engineering. In this study, we described a
CMCS scaffold containing controlled TGF-β1-releasing CSnp that may have practical
application to regenerative endodontics. The engineered matrix/nanoparticle scaffold
mimicked dental extracellular matrix, possessed high porosity, and significantly
enhanced SCAP migration and differentiation.
A properly engineered scaffold may serve as an initial attachment site for cells and BM
as well as structural support for immature tissue. Scaffolds in successful cell-free tissue
engineering applications should exhibit certain biophysical characteristics and deliver
BM to attract stem cells and promote their growth and differentiation. Scaffold porosity
facilitates diffusion of nutrients, clearance of degradation/cellular byproducts and cell
growth (45). The degree of crosslinking and porosity influences the rate of degradation of
a scaffold, which in turn, has been shown to affect the rate of release of immobilized
biomolecules over time (101, 143).
Scaffold degradation in vivo should be congruent with the new tissue growth, allowing
continued structural support and having little or no negative impact on the host tissue.
The impact of scaffold degradation byproducts on the residual functioning scaffold as
well as on the associated biological tissues deserves consideration. Undesirable evocation
of immune or inflammatory response, deterioration of scaffold characteristic properties
(e.g. stiffness, porosity, degradation rate), and host cell toxicity are legitimate concerns as
breakdown products of some materials have been shown to induce these negative effects
(49). Additionally, in the presence of infection, scaffold breakdown products should not
extend the inflammatory phase, which may delay healing.
The scaffolds used in this study consisted of chitosan and gelatin, two biopolymers
degraded by naturally occurring enzymes resident in human tissue, which show little to
28
no inflammatory response, and yield degradation products that include small sugar
molecules, peptides and amino acids well tolerated and processed by host cells (55, 137).
Moreover, chitosan demonstrates anti-bacterial and anti-inflammatory effects (74, 144).
The polycationic surface of chitosan allows adsorption of chitosan onto the anionic
bacterial cell surface, disrupting cell membrane integrity resulting in leakage of
intracellular components causing cell death. Chitosan also inhibits pro-inflammatory
cytokine production by host cells. It should be noted that changes in pH levels observed
during scaffold degradation in our study were negligible and remained at neutral levels.
Other scaffold materials have been shown to reduce the local tissue pH considerably (49).
Scaffolds fabricated by cross-linking gelatin with chitosan derivatives produced
improved mechanical properties and increased swelling capacity (137). Increases in
swelling capacity of the scaffold may be desirable for endodontic applications to improve
scaffold substantivity and adaptation to the root canal walls after implantation. Naturally,
concern must be directed to forces that may be exerted on the root wall and their potential
to initiate microcrack formation due to swelling of scaffold material placed inside the
root canal. The chitosan-gelatin scaffolds used in our study had dramatically low yield
strengths relative to dentin. Additionally, while swelling as a percentage of weight was
impressive in our study, the volumetric change was minimal and no significant increases
were detected at time points beyond 1 day. Furthermore, modifications to the scaffold
fabrication phase such as altering the gelatin content, scaffold porosity and degree of
crosslinking are examples of control over scaffold swelling and degradation to mitigate
this concern.
Favourable scaffold-cell interaction, as observed through SEM in our study, infers that
the CMCS scaffold used in this study was compatible with SCAP. SCAP are ecto-
mesenchymal stem cells from the apical papilla of developing human teeth and represent
a population of cells that can migrate to the root canal wall and participate in tissue
regeneration or organized repair. SCAP has demonstrated increased migration, tissue
regeneration capacity, and telomerase activity when compared to dental pulp stem cells
from the same tooth (120). It has also been suggested that SCAP may be better equipped
29
to survive and maintain their potential for differentiation in adverse, low oxygen
conditions (119).
TGF-β1 is a BM released from cells and incorporated into dentin and other non-
collagenous proteins in vivo. It has been identified as a promoter of odontogenic
differentiation (102, 103). However, availability of this molecule and other BM
sequestered in dentin matrix may not present in an optimum concentration or be available
for a duration necessary to satisfy stem cell requirements in tissue regeneration (139). In
the environment of a pulpless root canal, it is crucial that timely release of an optimum
concentration of growth factors be available for cells to survive, migrate and differentiate
(140). The importance of sustained release of BM for cell migration and differentiation
has previously been shown (113). Different concentrations of TGF-β1 were employed in
this study since it has been shown that TGF-β1 can effectively promote or inhibit cell
activity depending on the available concentration (100, 107). The significant increase in
SCAP viability compared to the same concentration of non-loaded CSnp or Free TGF-β1
was attributed to the release of bioactive TGF-β1 in a controlled manner over time. The
potential to provide such an environment was demonstrated in the TGF-β1-CSnp +
scaffold group where SCAP appeared to survive, migrate, and differentiate better than in
the other treatment groups.
TGF-β1 is known to modulate many cell activities including cell migration (140).
Significantly higher cell migration after 1 day in the Free TGF-β1 + scaffold group
compared with the TGF-β1-CSnp + scaffold group was expected since all of the TGF-β1
became available to the cells immediately when the TGF-β1 was in free form. After day
7, however, cell migration in the Free TGF-β1 + scaffold group decreased significantly,
which reflected a significant loss of bioactivity. In contrast, sustained release of TGF-β1
from TGF-β1-CSnp + scaffold group led to sustained cell migration over time. This was
an important finding in our study, as it showed the ability of the bioactive scaffold
containing TGF-β1-CSnp to address the issue of the short half-life of TGF-β1 (two to
three minutes) when it is used in free form in regenerative procedures (142).
30
Enhanced biomineralization of SCAP in the presence of TGF-β1-CSnp + scaffold was
also seen and can be attributed to the controlled release of TGF-β1 during the longer
periods of observation, as were the higher levels of DMP-1 and DSPP expression. DSPP
and DMP-1 are cytoplasmic proteins recognized as indicators of differentiation along
odontogenic lineages (145, 146). The immunofluorescent images also demonstrated more
uniform distribution of the cells in the TGF-β1-CSnp + scaffold group, when compared
to other experimental groups indicating that TGF-β1 released homogenously from the
CSnp dispersed in the scaffold.
In summary, biologically based regenerative endodontic procedures to address immature
teeth with pulp necrosis is a viable option. Increased success and outcome predictability
may be achievable by using a tissue engineering concept, which includes scaffolds, stem
cells, and bioactive molecules. Translatable findings using treatment strategies employing
materials that are biocompatible, anti-bacterial, anti-inflammatory and biodegradable may
offer another chairside option in the endodontic treatment of these teeth. Chitosan
appears to be an optimal material for this application due to its many positive attributes
previously described as well as its excellent functionality as a both a scaffold and
biomolecule delivery system as demonstrated in this study.
31
4.2 Conclusion In conclusion, the findings from the current study offered confirmation that CMCS
scaffold containing a sustained TGF-β1-releasing CSnp system:
• Possessed physicochemical characteristics conducive to SCAP survival and
attachment
• Promoted migration and enhanced differentiation of SCAP when compared to
Free TGF-β1 + scaffold, unloaded chitosan nanoparticles + scaffold, and scaffold
alone
• Illustrated the potential of chitosan-based scaffold to orchestrate BM delivery
with nanoparticle systems to facilitate dentin-pulp regeneration.
32
4.3 Future Directions Understanding both the role of individual BM and their reciprocal modulation in vivo will
be important to regenerative endodontics. Future research may include:
• Multiple BM release from polymeric scaffolds
• Modulation of scaffold/CSnp anti-bacterial/anti-inflammatory effect
• CSnp characterization over time in different tissue environments (e.g. pH,
temperature)
• Quality and spatial orientation of regenerated tissue
33
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Appendix I
0
200
400
600
800
1000
0 0.5 1 2 4 24
Swel
ling
ratio
(%)
Time (hours)
B A
0
10
20
30
40
50
60
0 7 14 21
Deg
rada
tion
(%)
Time (days)
C
D E
Figure 1. Characterization of scaffold: (A) Scaffold morphology as observed under SEM illustrating porous structure; (B) Scaffold swelling ratio; (C) Scaffold degradation (%) in the presence of lysozyme; (D) FTIR Spectra of CMCS, Gelatin, and the CMCS/gelatin scaffold; (E) SCAP morphology as observed under SEM illustrating favorable response to scaffold.
47
Figure 2: Characterization of TGF-β1-CSnp. (A) Cumulative release (ng) of TGF-β1 from TGF-β1-CSnp; (B) Relative viability of SCAP exposed to Free TGF-β1, TGF-β1-CSnp, or CSnp over 1, 3 and 14 days. (C) Representative fluorescent microscopic view after the calcein-AM staining (upper row, magnification 20X) and phase contrast microscopic images (lower row, magnification 40X). Data represent means ± SD (n = 3). *Significant difference between groups, p < 0.001.
0
20
40
60
80
100
120
140
1 3 14
Relative Viability (%
control)
Time (days)
B
Free TGF-‐b1
TGF-‐b1-‐CSnp
CSnp
Free TGF-β1
TGF-β1-CSnp CSnp
* *
0 2 4 6 8
10 12 14 16
0 100 200 300 400 500 600 700
Cum
ulat
ive
rele
ase
(ng)
Time (hours)
A
C
48
Figure 3: (A) TGF-β1-induced migration of SCAP: Cell migration was examined by a Transwell assay. SCAP were cultured in presence of Free TGF-β1, TGF-β1 released from TGF-β1-CSnp incubated for different time periods and CSnp. The lower membrane surfaces were photographed through a microscope at 20X magnification. Migrated cells in each field were counted. *Significant difference between groups, p < 0.028. (B) Effect of nanoparticles on biomineralization: ARS was quantified for biomineralization in SCAP culture for 3 weeks in presence of Free TGF-β1, TGF-β1-CSnp or CSnp. Data are shown as mean OD/µg of total protein. Data represent means ± SD (n = 3). *Significant difference between groups, p < 0.001.
0
0.001
0.002
0.003
0.004
0.005
0.006
1 week 2 week 3 week
OD
560/µ
g of
tot
al p
rote
in
Free TGF-b1
TGF-b1-CSnp
CSnp
B
7 14 21 Time (days)
Free TGF-β1 + scaffold TGF-β1-CSnp + scaffold
CSnp + scaffold
*
0
50
100
150
200
250
1 7 14 21
Mig
ratio
n (%
of c
ontr
ol)
Time (days)
Free TGF-b1
TGF-b1-CSnp
CSnp
A
Free TGF-β1 + scaffold TGF-β1-CSnp + scaffold
CSnp + scaffold
*
*
*
49
0
5000
10000
15000
20000
25000
Free TGF-‐β1 + scaffold TGF-‐β1-‐CSnp + scaffold CSnp + scaffold Scaffold
Relative Fluorescence
(arbitrary units)
DMP-‐1
DSPP
B * *
DMP-1 DSPP
A
Figure 4: Immunofluorescence of DMP-1 and DSPP during odontoblast differentiation: (A) FITC-conjugated antibody was used to detect the localization of the protein (green signal); all samples were counterstained with DAPI (blue signal). (B) Quantification of protein expression by ImageJ software and data are presented as relative mean fluorescence intensity. *Significant difference between groups, p < 0.001.